Changes in dorsomedial striatum activity during expression of goal-directed vs. habit-like cue-induced cocaine seeking.

A preclinical model of cue exposure therapy, cue extinction, reduces cue-induced cocaine seeking that is goal-directed but not habit-like. Goal-directed and habitual behaviors differentially rely on the dorsomedial striatum (DMS) and dorsolateral striatum (DLS), but the effects of cue extinction on dorsal striatal responses to cue-induced drug seeking are unknown. We used fiber photometry in rats trained to self-administer cocaine paired with an audiovisual cue to examine how dorsal striatal intracellular calcium and extracellular dopamine activity differs between goal-directed and habit-like cue-induced cocaine seeking and how it is impacted by cue extinction. After minimal fixed-ratio training, rats showed enhanced DMS and DLS calcium responses to cue-reinforced compared to unreinforced lever presses. After rats were trained on goal-promoting fixed ratio schedules or habit-promoting second-order schedules of reinforcement, different patterns of dorsal striatal calcium and dopamine responses to cue-reinforced lever presses emerged. Rats trained on habit-promoting second-order schedules showed reduced DMS calcium responses and enhanced DLS dopamine responses to cue-reinforced lever presses. Cue extinction reduced calcium responses during subsequent drug seeking in the DMS, but not in the DLS. Therefore, cue extinction may reduce goal-directed behavior through its effects on the DMS, whereas habit-like behavior and the DLS are unaffected.

Working memory performance predicts, but does not reduce, cocaine- and cannabinoid-seeking in adult male rats.

Background: Cognitive deficits reflecting impaired executive function are commonly associated with psychiatric disorders, including substance use. Cognitive training is proposed to improve treatment outcomes for these disorders by promoting neuroplasticity within the prefrontal cortex, enhancing executive control, and mitigating cognitive decline due to drug use. Additionally, brain derived neurotrophic factor (BDNF) can facilitate plasticity in the prefrontal cortex and reduce drug-seeking behaviors. We investigated whether working memory training could elevate BDNF levels in the prefrontal cortex and if this training would predict or protect against cocaine or cannabinoid seeking. Methods: Adult male rats were trained to perform a 'simple' or 'complex' version of a delayed- match-to-sample working memory task. Rats then self-administered cocaine or the synthetic cannabinoid WIN55,212-2 and were tested for cued drug-seeking during abstinence. Tissue from the prefrontal cortex and dorsal hippocampus was analyzed for BDNF protein expression. Results: Training on the working memory task enhanced endogenous BDNF protein levels in the prelimbic prefrontal cortex but not the dorsal hippocampus. Working memory training did not impact self-administration of either drug but predicted the extent of WIN self-administration and cocaine seeking during abstinence. Conclusions: These results suggest that working memory training promotes endogenous BDNF but does not alter drug-seeking or drug-taking behavior. However, individual differences in cognitive performance prior to drug exposure may predict vulnerability to future drug use.

Consequences of adolescent drug use.

Substance use in adolescence is a known risk factor for the development of neuropsychiatric and substance use disorders in adulthood. This is in part due to the fact that critical aspects of brain development occur during adolescence, which can be altered by drug use. Despite concerted efforts to educate youth about the potential negative consequences of substance use, initiation remains common amongst adolescents world-wide. Additionally, though there has been substantial research on the topic, many questions remain about the predictors and the consequences of adolescent drug use. In the following review, we will highlight some of the most recent literature on the neurobiological and behavioral effects of adolescent drug use in rodents, non-human primates, and humans, with a specific focus on alcohol, cannabis, nicotine, and the interactions between these substances. Overall, consumption of these substances during adolescence can produce long-lasting changes across a variety of structures and networks which can have enduring effects on behavior, emotion, and cognition.

The ventral tegmental area dopamine to lateral amygdala projection supports cocaine cue associative learning.

Learning and memory mechanisms are critically involved in drug craving and relapse. Environmental cues paired with repeated drug use acquire incentive value such that exposure to the cues alone can trigger craving and relapse. The amygdala, particularly the lateral amygdala (LA), underlies cue-related learning processes that assign valence to environmental stimuli including drug-paired cues. Evidence suggests that the ventral tegmental area (VTA) dopamine (DA) projection to the LA participates in encoding reinforcing effects that act as a US in conditioned cue reward-seeking as DA released in the amygdala is important for emotional and behavioral functions. Here we used chemogenetics to manipulate these VTA DA inputs to the LA to determine the role of this projection for acquisition of drug-cue associations and reinstatement of drug-seeking. We found inhibiting DA input to the LA during cocaine self-administration slowed acquisition and weakened the ability of the previously cocaine-paired cue to elicit cocaine-seeking. Conversely, exciting the projection during self-administration boosted the salience of the cocaine-paired cue as indicated by enhanced responding during cue-induced reinstatement. Importantly, interfering with DA input to the LA had no impact on the ability of cocaine to elicit a place preference or induce reinstatement in response to a priming cocaine injection. Overall, we show that manipulation of projections underlying DA signaling in the LA may be useful for developing therapeutic interventions for substance use disorders.

Changes in dorsomedial striatum activity mediate expression of goal-directed vs. habit-like cue-induced cocaine seeking.

A preclinical model of cue exposure therapy, cue extinction, reduces cue-induced cocaine seeking when drug seeking is goal-directed but not habitual. Goal-directed and habitual behaviors differentially rely on the dorsomedial striatum (DMS) and dorsolateral striatum (DLS), but the effects of cue extinction on dorsal striatal responses to cue-induced drug seeking are unknown. We used fiber photometry to examine how dorsal striatal intracellular calcium and extracellular dopamine activity differs between goal-directed and habitual cue-induced cocaine seeking and how it is impacted by cue extinction. Rats trained to self-administer cocaine paired with an audiovisual cue on schedules of reinforcement that promote goal-directed or habitual cocaine seeking had different patterns of dorsal striatal calcium and dopamine responses to cue-reinforced lever presses. Cue extinction reduced calcium and dopamine responses during subsequent drug seeking in the DMS, but not in the DLS. Therefore, cue extinction may reduce goal-directed behavior through its effects on the DMS, whereas habitual behavior and the DLS are unaffected.

Intermittent cocaine self-administration has sex-specific effects on addiction-like behaviors in rats.

Intermittent access (IntA) models of cocaine self-administration were developed to better model in rodents how cocaine is used by human drug users. Compared to traditional continuous access (ContA) models, IntA has been shown to enhance several pharmacological and behavioral effects of cocaine, but few studies have examined sex differences in IntA. Moreover, no one has examined the efficacy of cue extinction to reduce cocaine seeking in the IntA model, which has previously shown to be ineffective in other models that promote habit-like cocaine seeking. Therefore, rats were implanted with jugular vein catheters and dorsolateral striatum (DLS) cannulae and trained to self-administer cocaine paired with an audiovisual cue with ContA or IntA. In subsets of rats, we evaluated: the ability of Pavlovian cue extinction to reduce cue-induced drug seeking; motivation for cocaine using a progressive ratio procedure; punishment-resistant cocaine taking by pairing cocaine infusions with footshocks; and dependence of drug-seeking on DLS dopamine (a measure of habit-like behavior) with the dopamine antagonist cis-flupenthixol. Overall, cue extinction reduced cue-induced drug seeking after ContA or IntA. Compared to ContA, IntA resulted in increased motivation for cocaine exclusively in females, but IntA facilitated punished cocaine self-administration exclusively in males. After 10 days of IntA training, but not fewer, drug-seeking was dependent on DLS dopamine most notably in males. Our results suggest that IntA may be valuable for identifying sex differences in the early stages of drug use and provide a foundation for the investigation of the mechanisms involved.

Nicotine Enhances Intravenous Self-administration of Cannabinoids in Adult Rats.

INTRODUCTION: Nicotine and cannabis are commonly used together, yet few studies have investigated the effects of concurrent administration. Nicotine exhibits reinforcement enhancing effects by promoting the reinforcing properties of stimuli including other drugs. As many studies of this effect used non-contingent nicotine, we implemented a dual-self-administration model where rats have simultaneous access to two drugs and choose which to self-administer throughout a session. Here, we investigated the effect of self-administered or non-contingently delivered nicotine on cannabinoid self-administration. METHODS: Adult male rats were allowed to self-administer the synthetic cannabinoid WIN 55,212-2 (WIN) intravenously, with or without subcutaneous nicotine injections before each session. A separate group of animals were allowed to self-administer WIN, nicotine, or saline using a dual-catheter procedure, where each solution was infused independently and associated with a separate operant response. A third group of male and female rats were allowed to self-administer delta-9-tetrahydrocannabinol (THC) with or without pre-session injections of nicotine. RESULTS: Nicotine injections increased self-administration of WIN and THC. During dual self-administration, nicotine availability increased saline and WIN infusions but nicotine intake was not changed by WIN or saline availability. Rats preferred nicotine over saline, but preferred nicotine and WIN equally when both were available. The effect of nicotine on cannabinoid self-administration was acute and reversible when nicotine was no longer present. CONCLUSIONS: These results expand our understanding of the ability of nicotine to enhance reinforcement of other drugs and suggest that co-use of nicotine and cannabinoids promotes cannabinoid use beyond what would be taken alone. IMPLICATIONS: This study utilizes a dual intravenous self-administration model to investigate the ability of nicotine to enhance cannabinoid intake. Our results demonstrate that the reinforcement enhancing properties of nicotine on drug use extend to include cannabinoids, but that this effect occurs specifically when nicotine is administered alongside the cannabinoid. Interestingly, cannabinoid use did not promote nicotine intake, suggesting this mechanism of reinforcement is specific to nicotine.

Sex- and age-dependent effects of chronic corticosterone exposure on depressive-like, anxiety-like, and fear-related behavior: Role of amygdala glutamate receptors in the rat.

Persistent glucocorticoid elevation consistent with chronic stress exposure can lead to psychopathology, including mood and anxiety disorders. Women and stress-exposed adolescents are more likely to be diagnosed with mood disorders, suggesting that sex and age are important factors in determining vulnerability, though much remains to be determined regarding the mechanisms underlying this risk. Thus, the aim of the present experiments was to use the chronic corticosterone (CORT) exposure paradigm, a model of depression-like behavior that has previously been established primarily in adult males, to determine the mood-related effects of CORT in female and adolescent rats. Depression- and anxiety-like effects in adulthood were determined using the sucrose preference (SPT), the forced swim test (FST), the elevated plus maze, and fear conditioning. Basolateral amygdala (BLA) and medial prefrontal cortex (mPFC) glutamate receptor subunit levels were then measured. In a subsequent experiment, adult male and female rats were tested for the effects of pharmacological activation (via AMPA) or inhibition (via NBQX) of AMPA receptors in the BLA on behavior in the FST. Overall, females showed reduced anxiety- and depressive-like behaviors relative to males. However, females treated with CORT in adolescence, but not adulthood, had increased immobility in the FST, indicative of depression-like behavior. In contrast, CORT did not alter behavior in adolescent-treated males, though the previously reported depression-like effect of adult CORT exposure was observed. Control females had higher expression of the AMPA receptor subunits GluA1 and GluA2/3 selectively in the BLA relative to males. Adolescent CORT treatment, however, decreased BLA GluA1 and GluA2/3 expression in females, but increased expression in males, consistent with the direction of depression-like behavioral effects. Male and female rats also demonstrated opposing patterns of response to BLA AMPA receptor modulation in the FST, with AMPA infusion magnifying the sex difference of decreased immobility in females. Overall, these experiments show that increased glutamate receptor function in the BLA may decrease the risk of developing depressive-like behavior, further supporting efforts to target glutamatergic receptors for the treatment of stress-related psychiatric disorders. These findings also support further focus on sex as a biological variable in neuropsychiatric research.

Using Optogenetics to Reverse Neuroplasticity and Inhibit Cocaine Seeking in Rats.

This protocol demonstrates the steps needed to use optogenetic tools to reverse cocaine-induced plasticity at thalamo-amygdala circuits to reduce subsequent cocaine seeking behaviors in the rat. In our research, we had found that when rats self-administer intravenous cocaine paired with an audiovisual cue, synapses formed at inputs from the medial geniculate nucleus of the thalamus (MGN) onto principal neurons of the lateral amygdala (LA) become stronger as the cue-cocaine association is learned. We hypothesized that reversal of the cocaine-induced plasticity at these synapses would reduce cue-motivated cocaine seeking behavior. In order to accomplish this type of neuromodulation in vivo, we wanted to induce synaptic long-term depression (LTD), which decreases the strength of MGN-LA synapses. To this end, we used optogenetics, which allows neuromodulation of brain circuits using light. The excitatory opsin oChiEF was expressed on presynaptic MGN terminals in the LA by infusing an AAV containing oChiEF into the MGN. Optical fibers were then implanted in the LA and 473 nm laser light was pulsed at a frequency of 1 Hz for 15 minutes to induce LTD and reverse cocaine induced plasticity. This manipulation produces a long-lasting reduction in the ability of cues associated with cocaine to induce drug seeking actions.

Valence encoding in the amygdala influences motivated behavior.

The amygdala is critical for emotional processing and motivated behavior. Its role in these functions is due to its processing of the valence of environmental stimuli. The amygdala receives direct sensory input from sensory thalamus and cortical regions to integrate sensory information from the environment with aversive and/or appetitive outcomes. As many reviews have discussed the amygdala's role in threat processing and fear conditioning, this review will focus on how the amygdala encodes positive valence and the mechanisms that allow it to distinguish between stimuli of positive and negative valence. These findings are also extended to consider how valence encoding populations in the amygdala contribute to local and long-range circuits including those that integrate environmental cues and positive valence. Understanding the complexity of valence encoding in the amygdala is crucial as these mechanisms are implicated in a variety of disease states including anxiety disorders and substance use disorders.

Roles of dopamine and glutamate co-release in the nucleus accumbens in mediating the actions of drugs of abuse.

Projections of ventral tegmental area dopamine (DA) neurons to the medial shell of the nucleus accumbens have been increasingly implicated as integral to the behavioral and physiological changes involved in the development of substance use disorders (SUDs). Recently, many of these nucleus accumbens-projecting DA neurons were found to also release the neurotransmitter glutamate. This glutamate co-release from DA neurons is critical in mediating the effect of drugs of abuse on addiction-related behaviors. Potential mechanisms underlying the role(s) of dopamine/glutamate co-release in contributing to SUDs are unclear. Nevertheless, an important clue may relate to glutamate's ability to potentiate loading of DA into synaptic vesicles within terminals in the nucleus accumbens in response to drug-induced elevations in neuronal activity, enabling a more robust release of DA after stimulation. Here, we summarize how drugs of abuse, particularly cocaine, opioids, and alcohol, alter DA release in the nucleus accumbens medial shell, examine the potential role of DA/glutamate co-release in mediating these effects, and discuss future directions for further investigating these mechanisms.

Dorsolateral striatum dopamine-dependent cocaine seeking is resistant to pavlovian cue extinction in male and female rats.

Cue exposure therapy (CET) reduces craving induced by drug-associated cues in individuals with substance use disorders. A preclinical model of CET, cue extinction, similarly reduces cue-induced cocaine seeking in rodent self-administration models; however, those models may not capture the habitual or compulsive aspects of drug use. Thus, the effectiveness of cue extinction was tested in male and female rats trained to self-administer cocaine using second-order (SO) or fixed-ratio (FR) schedules of reinforcement to facilitate dorsolateral striatum (DLS) dopamine-dependent or -independent response strategies, respectively. Cue extinction significantly reduced drug seeking in FR-trained rats, replicating prior results, but was ineffective in SO-trained rats. SO-trained rats also showed increased indices of glutamate signaling in the DLS relative to FR-trained rats, despite comparable levels of cocaine intake. Furthermore, in SO-trained rats, antagonism of AMPA receptors in the DLS rescued the efficacy of cue extinction to reduce drug seeking. Finally, the effectiveness of cue extinction was also revealed in SO-trained rats when they were taught to perform a new, non-habitual response for cocaine cue presentation. Overall, our findings indicate that habit-like drug seeking leads to plasticity in the DLS and behavior that is resistant to cue extinction, but that the effects of cue extinction are restored when DLS glutamatergic signaling is blocked. These results have implications for the effectiveness of clinical cue exposure therapy.

Intravenous self-administration of delta-9-THC in adolescent rats produces long-lasting alterations in behavior and receptor protein expression.

RATIONALE: Initial exposure to cannabinoids, including Δ-9-tetrahydrocannabinol (THC), often occurs during adolescence. Considerable neurodevelopmental alterations occur throughout adolescence, and the environmental insult posed by exogenous cannabinoid exposure may alter natural developmental trajectories. Multiple studies suggest that long-lasting deficits in cognitive function occur as a result of adolescent cannabis use, but considerable variability exists in the magnitude of these effects. OBJECTIVES: We sought to establish a novel procedure for achieving intravenous THC self-administration in adolescent rats in order to determine if volitional THC intake in adolescence produced indices of addiction-related behavior, altered working memory performance in adulthood, or altered the expression of proteins associated with these behaviors across several brain regions. METHODS: Male and female adolescent rats learned to operantly self-administer escalating doses of THC intravenously from PD 32-51. Upon reaching adulthood they were tested in abstinence for cued reinstatement of THC-seeking and working memory performance on a delayed-match-to-sample task. In a separate cohort, glutamatergic, GABAergic, and cannabinoid receptor protein expression was measured in multiple brain regions. RESULTS: Both male and female adolescents self-administered THC and exhibited cue-induced lever pressing throughout abstinence. THC-exposed males exhibited slightly enhanced working memory performance in adulthood, and better performance positively correlated with total THC self-administered during adolescence. Adolescent THC-exposed rats exhibited reductions in CB1, GABA, and glutamate receptor protein, primarily in the prefrontal cortex, dorsal hippocampus, and ventral tegmental area. CONCLUSIONS: These results suggest that THC exposure at self-administered doses can produce moderate behavioral and molecular alterations, including sex-dependent effects on working memory performance in adulthood.

Disentangling the lasting effects of adolescent cannabinoid exposure.

Cannabis is the most widely used illicit substance among adolescents, and adolescent cannabis use is associated with various neurocognitive deficits that can extend into adulthood. A growing body of evidence supports the hypothesis that adolescence encompasses a vulnerable period of development where exposure to exogenous cannabinoids can alter the normative trajectory of brain maturation. In this review, we present an overview of studies of human and rodent models that examine lasting effects of adolescent exposure. We include evidence from meta-analyses, longitudinal, or cross-sectional studies in humans that consider age of onset as a factor that contributes to the behavioral dysregulation and altered structural or functional development in cannabis users. We also discuss evidence from preclinical rodent models utilizing well-characterized or innovative routes of exposure, investigating the effects of dose and timing to produce behavioral deficits or alterations on a neuronal and behavioral level. Multiple studies from both humans and animals provide contrasting results regarding the magnitude of residual effects. Combined evidence suggests that exposure to psychoactive cannabinoids during adolescence has the potential to produce subtle, but lasting, alterations in neurobiology and behavior.

Molecular and circuit mechanisms regulating cocaine memory.

Risk of relapse is a major challenge in the treatment of substance use disorders. Several types of learning and memory mechanisms are involved in substance use and have implications for relapse. Associative memories form between the effects of drugs and the surrounding environmental stimuli, and exposure to these stimuli during abstinence causes stress and triggers drug craving, which can lead to relapse. Understanding the neural underpinnings of how these associations are formed and maintained will inform future advances in treatment practices. A large body of research has expanded our knowledge of how associative memories are acquired and consolidated, how they are updated through reactivation and reconsolidation, and how competing extinction memories are formed. This review will focus on the vast literature examining the mechanisms of cocaine Pavlovian associative memories with an emphasis on the molecular memory mechanisms and circuits involved in the consolidation, reconsolidation, and extinction of these memories. Additional research elucidating the specific signaling pathways, mechanisms of synaptic plasticity, and epigenetic regulation of gene expression in the circuits involved in associative learning will reveal more distinctions between consolidation, reconsolidation, and extinction learning that can be applied to the treatment of substance use disorders.

Calcineurin Promotes Neuroplastic Changes in the Amygdala Associated with Weakened Cocaine-Cue Memories.

Interfering with memory reconsolidation or inducing memory extinction are two approaches for weakening maladaptive memories in disorders such as addiction and post-traumatic stress disorder. Both extinction and reconsolidation are regulated by intracellular protein kinases and phosphatases, and interfering with these signaling molecules can alter memory strength. The calcium-dependent protein phosphatase, calcineurin (CaN), has been implicated in both the consolidation and extinction of fear memories. However, the role of CaN in regulating drug-cue associative memories has not been investigated. Prior studies have demonstrated that plasticity at thalamo-lateral amygdala (T-LA) synapses is critically involved in the regulation of cocaine-cue memories. Therefore, in the present study, we tested the effects of LA administration of an activator of CaN, chlorogenic acid (CGA), on behavioral and electrophysiological indices of cocaine cue memory reconsolidation and extinction. Male, Sprague-Dawley rats were trained to self-administer cocaine paired with an audiovisual cue. The cue memory was then either briefly reactivated, extinguished, or not manipulated, followed immediately by LA infusion of CGA. Rats were tested 24 h later for cue-induced reinstatement, or LA slices were prepared for electrophysiological recordings. We found that intra-LA infusions of CGA following cue extinction or reconsolidation reduced cue-induced reinstatement, which was blocked by co-infusion of the CaN inhibitor, FK-506. Similarly, CGA infusions following cue re-exposure significantly attenuated EPSC amplitude at T-LA synapses, suggesting that CaN affects cocaine-cue memory reconsolidation and extinction by altering T-LA synaptic strength. Therefore, CaN signaling in the LA may represent a novel target for disrupting cocaine-associated memories to reduce relapse.SIGNIFICANCE STATEMENT Repetitive drug use induces synaptic plasticity that underlies the formation of long-lasting associative memories for environmental cues paired with the drug. We previously identified thalamo-amygdala synapses (T-LA) that project via the interal capsule, as an important locus for the regulation of cocaine-cue memories. These synapses are strengthened by repeated cocaine-cue pairings, but this is reversed by extinction training or by optogenetic induction of in vivo long-term depression (LTD). Here, we demonstrate that activating calcineurin, a calcium-dependent phosphatase, following the reactivation or extinction of a cocaine-cue memory, induces LTD-like changes at T-LA synapses, and a corresponding decrease in cue-induced reinstatement, suggesting that calcineurin may be a potential therapeutic target for relapse prevention.

Estradiol modulation of the renin-angiotensin system and the regulation of fear extinction.

Post-traumatic stress disorder (PTSD) is more prevalent in women than men, yet much remains to be determined regarding the mechanism underlying this sex difference. Clinical and preclinical studies have shown that low estradiol levels during extinction of fear conditioning in rodents (i.e., cue exposure therapy in humans) leads to poor extinction consolidation and increased fear during extinction recall. The renin-angiotensin system (RAS) is also associated with stress-related pathologies, and RAS antagonists can enhance extinction consolidation in males. However, less is known about how estradiol and the RAS converge to alter fear extinction consolidation in females. Since estradiol downregulates the RAS, we determined the role of surgically (via ovariectomy [OVX]) and pharmacologically (via the hormonal contraceptive [HC], levonorgestrel) clamping estradiol at low levels in female rats on fear-related behavior, serum estradiol and angiotensin II (Ang II) levels, and angiotensin II type I receptor (AT1R) binding in the brain. We then tested whether the AT1R antagonist losartan would alter fear-related behavior in an estradiol-dependent manner. We found that both OVX and HC treatment produced extinction consolidation deficits relative to intact female rats in proestrus (when estradiol levels are high), and that losartan treatment mitigated these deficits and reduced freezing. OVX, but not HC, altered AT1R ligand binding, though HC reduced estradiol and increased Ang II levels in plasma. These findings have significant clinical implications, indicating that administration of an AT1R antagonist, especially if estradiol levels are low, prior to an exposure therapy session may improve treatment outcomes in females.

Plasticity at Thalamo-amygdala Synapses Regulates Cocaine-Cue Memory Formation and Extinction.

Repeated drug use has long-lasting effects on plasticity throughout the brain's reward and memory systems. Environmental cues that are associated with drugs of abuse can elicit craving and relapse, but the neural circuits responsible for driving drug-cue-related behaviors have not been well delineated, creating a hurdle for the development of effective relapse prevention therapies. In this study, we used a cocaine+cue self-administration paradigm followed by cue re-exposure to establish that the strength of the drug cue association corresponds to the strength of synapses between the medial geniculate nucleus (MGN) of the thalamus and the lateral amygdala (LA). Furthermore, we demonstrate, via optogenetically induced LTD of MGN-LA synapses, that reversing cocaine-induced potentiation of this pathway is sufficient to inhibit cue-induced relapse-like behavior.